CD24 is highly useful in differentiating high-grade serous carcinoma from benign and malignant mesothelial cells

doi:10.1016/j.humpath.2016.08.005

Human Pathology

Volume 58, December 2016, Pages 123-127

https://doi.org/10.1016/j.humpath.2016.08.005 Get rights and content

Summary

CD24 was previously shown to be overexpressed in high-grade serous carcinoma (HGSC) effusions compared to malignant mesothelioma (MM) in gene expression array analysis. The present study validated this observation in a large series consisting of both effusions and surgical specimens. Effusions (n = 206; 100 HGSC, 16 ovarian carcinomas of other histological types, 54 breast carcinomas, 36 MM) and surgical specimens (n = 182; 117 ovarian carcinomas, 65 MM) were analyzed for CD24 expression using immunohistochemistry. CD24 was expressed in 105/116 (91%) ovarian carcinoma and 16/54 (30%) breast carcinoma effusions, while it was uniformly absent in MM (0/36; 0%; P < .001). Reactive mesothelial cells were CD24-negative in all carcinoma specimens. Comparative analysis of 117 solid primary (n = 43) and metastatic (n = 74) ovarian carcinomas and 65 solid MM specimens showed CD24 expression in 46% (54/117) of the former compared to 3% (2/65) of the latter (P < .001). Comparative analysis of ovarian carcinomas at different anatomic sites showed significantly higher CD24 expression in effusions compared to solid ovarian and metastatic lesions (P < .001), with similar results when analysis was limited to HGSC (P < .001). In conclusion, CD24 is a highly sensitive and specific marker of ovarian carcinoma in the differential diagnosis from MM and reactive mesothelium in effusions. CD24 is similarly a specific marker in surgical specimens, though with lower sensitivity. The overexpression of CD24 in ovarian carcinoma effusions compared to solid lesions may be due to the acquisition of cancer stem cell characteristics.

Introduction

Ovarian cancer, consisting predominantly of ovarian carcinoma (OC), is the most lethal gynecological cancer. Efforts directed at improving the outcome of OC patients have been checked by the absence of a robust test for early detection, as well as by chemoresistance that develops in the majority of patients after repeated cycles of chemotherapy. Targeted therapy has to date shown only modest effect in changing this bleak picture [1], [2].

It is widely recognized that small tumor cell populations escape death by chemotherapy and constitute the cellular niche from which recurrence develops in many cancers. The term cancer stem cells (CSC) has been used by some investigators to describe these cells and different markers, including ALDH1A1, CD44, CD117 (c-Kit), CD133 and EpCAM, have been proposed as putative CSC markers in OC (reviewed by Jaggupili et al, Burgos-Ojeda et al, Garson et al [3], [4], [5]).

While the validity of the above-mentioned proteins as CSC markers has been questioned, the case is still more ambiguous for CD24, a cell surface glycoprotein mediating tumor cell adhesion to P-selectin expressed on endothelial cells and platelets, and thus postulated to promote metastasis [5]. CD24 has been described as both positive and negative CSC marker, partly depending on tumor origin, but also in OC itself [3].

Immunohistochemistry (IHC) is the most widely used ancillary method in both cytopathology and surgical pathology, and panels which aid in the detection and classification of cancers involving the serosal cavities have been constantly expanding and improving in recent years. The differential diagnosis between serous carcinoma of female genital origin and malignant mesothelioma (MM) is nevertheless difficult in some cases owing to the overlapping morphology and immunomarker expression of these tumors. The majority of antibodies are not 100% sensitive or specific, and the use of several markers is therefore mandated [6].

CD24 was previously identified by our group as a specific marker of ovarian/peritoneal serous carcinoma in the differential diagnosis from diffuse malignant peritoneal mesothelioma (DMPM) using gene expression analysis applied to effusion specimens [7]. Results were not validated at the protein level in the original study, nor has this observation been followed by other investigators to the best of the author's knowledge. The present study analyzed the diagnostic role of CD24 in differentiating OC, primarily high-grade serous carcinoma (HGSC), from MM.

Section snippets

Effusion specimens

Effusions consisted of 116 and 36 specimens from patients diagnosed with OC and MM, respectively. Additionally, 54 breast carcinoma (BC) effusions were included for comparative purposes (total = 206). Effusions were submitted to the Department of Pathology at the Norwegian Radium Hospital for routine diagnostic purposes during the period 1998–2008, with the exception of 8 MM specimens from the Department of Pathology in Aalborg, Denmark, used with kind permission from Mr. Søren Nielsen.

Results

CD24 expression results are detailed in the Table and representative specimens are shown in Figure. Staining was localized to the membrane. CD24 was expressed in 105/116 (91%) OC and was present in both HGSC and tumors of other histology, compared to 16/54 (30%) breast carcinomas. CD24 staining was uniformly absent in MM effusions (0/36; 0%). Differences between the 3 tumor types were significant in both 3-tier analysis and in separate comparison of HGSC versus MM and HGSC versus breast

Discussion

An IHC panel applied to the diagnosis of serosal cancers should ideally consist of epithelial and mesothelial markers with high specificity and sensitivity, which allow for a certain diagnosis of carcinoma, MM or RMC using a limited antibody panel. While a large number of markers differentiating MM from metastatic carcinoma have been described, few of them are useful in the differential diagnosis of HGSC from MM. MM-related proteins such as mesothelin, WT1, podoplanin/D2-40 and cytokeratin 5/6

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Cited by (27)

ALDH1A1+ ovarian cancer stem cells co-expressing surface markers CD24, EPHA1 and CD9 form tumours in vivo
2020, Experimental Cell Research
One of the reasons for recurrence following treatment of high grade serous ovarian carcinoma (HGSOC) is the persistence of residual cancer stem cells (CSCs). There has been variability between laboratories in the identification of CSC markers for HGSOC. We have identified new surface markers (CD24, CD9 and EPHA1) in addition to those previously known (CD44, CD117 and CD133) using a bioinformatics approach. The expression of these surface markers was evaluated in ovarian cancer cell lines, primary malignant cells (PMCs), normal ovary and HGSOC. There was no preferential expression of any of the markers or a combination. All the markers were expressed at variable levels in ovarian cancer cell lines and PMCs. Only CD117 and CD9 were expressed in the normal ovarian surface epithelium and fallopian tube. Both ALDEFLUOR (ALDH1A1) and side population assays identified a small proportion of cells (<3%) separately that did not overlap with little variability in cell lines and PMCs. All surface markers were co-expressed in ALDH1A1+ cells without preference for one combination. The cell cycle analysis of ALDH1A1+ cells alone revealed that majority of them reside in G0/G1 phase of cell cycle. Further separation of G0 and G1 phases showed that ALDH1A1+ cells reside in G1 phase of the cell cycle. Xenograft assays showed that the combinations of ALDH1A1 + cells co-expressing CD9, CD24 or EPHA1 were more tumorigenic and aggressive with respect to ALDH1A1-cells. These data suggest that a combined approach could be more useful in identifying CSCs in HGSOC.
CD24-targeted intraoperative fluorescence image-guided surgery leads to improved cytoreduction of ovarian cancer in a preclinical orthotopic surgical model
2020, EBioMedicine
The completeness of resection is a key prognostic indicator in patients with ovarian cancer, and the application of tumour-targeted fluorescence image-guided surgery (FIGS) has led to improved detection of peritoneal metastases during cytoreductive surgery. CD24 is highly expressed in ovarian cancer and has been shown to be a suitable biomarker for tumour-targeted imaging.
CD24 expression was investigated in cell lines and heterogenous patient-derived xenograft (PDX) tumour samples of high-grade serous ovarian carcinoma (HGSOC). After conjugation of the monoclonal antibody CD24 to the NIR dye Alexa Fluor 750 and the evaluation of the optimal pharmacological parameters (OV-90, n = 21), orthotopic HGSOC metastatic xenografts (OV-90, n = 16) underwent cytoreductive surgery with real-time feedback. The impact of intraoperative CD24-targeted fluorescence guidance was compared to white light and palpation alone, and the recurrence of disease was monitored post-operatively (OV-90, n = 12). CD24-AF750 was further evaluated in four clinically annotated orthotopic PDX models of metastatic HGSOC, to validate the translational potential for intraoperative guidance.
CD24-targeted intraoperative NIR FIGS significantly (47•3%) improved tumour detection and resection, and reduced the post-operative tumour burden compared to standard white-light surgery in orthotopic HGSOC xenografts. CD24-AF750 allowed identification of minuscule tumour lesions which were undetectable with the naked eye in four HGSOC PDX.
CD24-targeted FIGS has translational potential as an aid to improve debulking surgery of ovarian cancer.
This study was supported by the H2020 program MSCA-ITN [675743], Helse Vest RHF, and Helse Bergen HF [911809, 911852, 912171, 240222, 911974, HV1269], as well as by The Norwegian Cancer Society [182735], and The Research Council of Norway through its Centres of excellence funding scheme [223250, 262652].
CD24-targeted fluorescence imaging in patient-derived xenograft models of high-grade serous ovarian carcinoma: Fluorescence imaging of PDX models of HGSOC
2020, EBioMedicine
Citation Excerpt :
The cell surface marker CD24 has been shown to be significantly upregulated in a number of carcinomas compared to their benign counterparts, suggesting it as an ideal target for theranostic applications [44-46]. In our cohort of 30 cases of epithelial ovarian carcinoma we found expression of CD24 present in 90% of tissues, concurring with the observation that CD24 is highly expressed in the vast majority of ovarian cancer cases [47-49] with highest expression levels in the serous subtype [47,48,50,51]. Overdevest et al. reported higher CD24 staining indices in metastases and identified CD24 as the most overexpressed amongst transcripts in metastasising cells of human bladder carcinoma [52].
The survival rate of patients with advanced high-grade serous ovarian carcinoma (HGSOC) remains disappointing. Clinically translatable orthotopic cell line xenograft models and patient-derived xenografts (PDXs) may aid the implementation of more personalised treatment approaches. Although orthotopic PDX reflecting heterogeneous molecular subtypes are considered the most relevant preclinical models, their use in therapeutic development is limited by lack of appropriate imaging modalities.
We developed novel orthotopic xenograft and PDX models for HGSOC, and applied a near-infrared fluorescently labelled monoclonal antibody targeting the cell surface antigen CD24 for non-invasive molecular imaging of epithelial ovarian cancer. CD24-Alexa Fluor 680 fluorescence imaging was compared to bioluminescence imaging in three orthotopic cell line xenograft models of ovarian cancer (OV-90^luc+, Skov-3^luc+ and Caov-3^luc+, n = 3 per model). The application of fluorescence imaging to assess treatment efficacy was performed in carboplatin-paclitaxel treated orthotopic OV-90 xenografts (n = 10), before the probe was evaluated to detect disease progression in heterogenous PDX models (n = 7).
Application of the near-infrared probe, CD24-AF680, enabled both spatio-temporal visualisation of tumour development, and longitudinal therapy monitoring of orthotopic xenografts. Notably, CD24-AF680 facilitated imaging of multiple PDX models representing different histological subtypes of the disease.
The combined implementation of CD24-AF680 and orthotopic PDX models creates a state-of-the-art preclinical platform which will impact the identification and validation of new targeted therapies, fluorescence image-guided surgery, and ultimately the outcome for HGSOC patients.
This study was supported by the H2020 program MSCA-ITN [675743], Helse Vest RHF, and Helse Bergen HF [911809, 911852, 912171, 240222, HV1269], as well as by The Norwegian Cancer Society [182735], and The Research Council of Norway through its Centers of excellence funding scheme [223250, 262652].
Expression of CD24 in plasma, exosome and ovarian tissue samples of serous ovarian cancer patients
2019, Journal of Biotechnology
CD24 is a small molecular weight cell-surface protein and an independent marker for poor prognosis in the different type of cancers. We aimed to determine the expression of CD24 in plasma, exosomes and ovarian tissue samples of serous ovarian cancer patients.
We collected tissue and blood samples from 21 cases of serous ovarian cancer and eight healthy controls. We used silica adsorption method for isolation of RNA. The cDNA was synthesized using quantitative real-time PCR. We used beta-globin as a housekeeping gene for the normalization of the data. Protein-protein and miRNA networking were analyzed.
There was a significant difference in the expression of CD24 in ovarian tissue between controls and patients (0.16 ± 0.32 vs. 44.97 ± 68.06; p < 0.01), while CD24 did not show expression in each plasma and exosome samples. There was a correlation in the expression of CD24 and FIGO grading between controls and patients.
CD24 expression was detected in exosomes in 38.1% of patients, mainly with FIGO III, and in their plasma in 9.5% of cases. Our network analysis shows LYN, SELP, FGR, and NPM1 proteins are interacting with CD24.
Our study demonstrates higher expression of CD24 in ovarian cancer patients’ tissue samples, and there is an association with FIGO classification. However, CD24 showed expression only in some cell-free plasma and exosome samples.
SOX2 and SOX9 are markers of clinically aggressive disease in metastatic high-grade serous carcinoma
2019, Gynecologic Oncology
Citation Excerpt :
However, the clinical relevance of these markers has not been assessed in large series of patients with OC effusions. Two recent studies by a member of our group have failed to identify such role for CD24 and nestin in this anatomic compartment [21,22]. The present study assessed the clinical and biological role of the CSC markers in HGSC.
The aim of this study was to analyze the expression, biological role and clinical relevance of cancer stem cell markers in high-grade serous carcinoma (HGSC).
mRNA expression by qRT-PCR of NANOG, OCT4, SOX2, SOX4, SOX9, LIN28A and LIN28B was analyzed in 134 HGSC specimens (84 effusions, 50 surgical specimens). Nanog, OCT3/4, SOX2 and SOX9 protein expression by immunohistochemistry was analyzed in 52 HGSC effusions. Nanog protein expression in exosomes from 80 HGSC effusions was studied by Western Blotting. OVCAR3 cells underwent CRISPR/Cas9 Nanog knockout (KO), and the effect of Nanog KO on migration, invasion, proliferation and proteolytic activity was analyzed in OVCAR3 and OVCAR8 cells.
OCT4 mRNA was overexpressed in effusions compared to solid specimens (p = 0.046), whereas SOX9 was overexpressed in the ovarian tumors compared to effusions and solid metastases (p = 0.003). Higher SOX2 and SOX9 expression was associated with primary (intrinsic) chemoresistance (p = 0.009 and p = 0.02, respectively). Higher SOX9 levels were associated with shorter overall survival in univariate (p = 0.04) and multivariate (p = 0.049) analysis. OCT3/4, SOX2 and SOX9 proteins were found in HGSC cells, whereas Nanog was detected only in exosomes. Higher SOX2 protein expression was associated with shorter overall survival in univariate analysis (p = 0.049). OVCAR cells exposed to OVCAR3 NANOG KO exosomes had reduced migration, invasion and MMP9 activity.
SOX2 and SOX9 mRNA levels in HGSC effusions may be markers of clinically aggressive disease. Nanog is secreted in HGSC exosomes in effusions and modulates tumor-promoting cellular processes in vitro.
Ovarian cancer stem cells and their role in drug resistance
2019, International Journal of Biochemistry and Cell Biology
Citation Excerpt :
Their relationship to OC chemo-resistance and use as potential targets for therapies is discussed below. CD24 (Cluster of Differentiation 24) is a 27–30 amino acid cell surface protein anchored to the cell membrane by glycosyl-phosphotidyl-inositol, linked to a wide variety of cancers, such as breast, bladder, colorectal and OC (Kristiansen et al., 2004; Nakamura et al., 2017; Davidson, 2016; Jaggupilli and Elkord, 2012). CD24 is thought to increase metastasis by promoting the adhesion of tumour cells to P-selectin, an adhesion receptor found on platelets and endothelial cells.
Ovarian cancer is typically diagnosed at advanced stages (III or IV), with metastasis ensuing at stage III. Complete remission is infrequent and is not achieved in almost half of the women diagnosed with ovarian cancer. Consequently, management and treatment of this disease is challenging as many patients are faced with tumour recurrence disseminating to surrounding organs further complicated with acquired chemo-resistance. The cancer stem cell theory proposes the idea that a drug resistant subset of tumour cells drive tumour progression, metastasis and ultimately, recurrent disease. In the ovarian cancer field, cancer stem cells remain elusive with significant gaps in our knowledge. The characteristics and specific role of ovarian cancer stem cells in recurrence still requires further research since different studies often arrive at contradictory conclusions. Here we present a review and critical analysis of current research conducted in the field of ovarian cancer stem cells and their potential role in drug resistance including several signalling pathways within these cells that affect the viability of targeted therapies.

View all citing articles on Scopus

^☆: Competing interests: None declared.

^☆☆: Funding/Support: This work was supported by Inger and John Fredriksen Foundation for Ovarian Cancer Research.

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Original contributionCD24 is highly useful in differentiating high-grade serous carcinoma from benign and malignant mesothelial cells☆,☆☆

Summary

Introduction

Section snippets

Effusion specimens

Results

Discussion

Cancer Lett

Hum Pathol

Hum Pathol

Mod Pathol

Mod Pathol

Hum Pathol

Hum Pathol

Am J Pathol

Gynecol Oncol

Ovarian cancer - diagnostic, biological and prognostic aspects

Womens Health

Recently identified drug resistance biomarkers in ovarian cancer

Expert Rev Mol Diagn

Significance of CD44 and CD24 as cancer stem cell markers: an enduring ambiguity

Clin Dev Immunol

Original contribution
CD24 is highly useful in differentiating high-grade serous carcinoma from benign and malignant mesothelial cells☆,☆☆